title: Demonstration of at least two different actin-binding sites in villin, a calcium-regulated modulator of F-actin organization.
abstract: Villin, one of the calcium regulated modulator proteins of F-actin organization, restricts F-actin to short filaments in the presence of calcium and bundles F-actin in the absence of calcium. Limited in vitro proteolysis of villin generates, in addition to a large core fragment (apparent Mr = 90,000) previously described, a small headpiece (Mr = 8,500). The finding that the F-actin nucleation and severing activity of villin, but not its bundling activity, is retained by the core suggested that the headpiece may be directly involved in bundling. Headpiece has now been purified and characterized. It shows strong F-actin binding both in the presence and absence of calcium, leading to a final stoichiometry of 1 headpiece to 1 F-actin monomer. Headpiece also inhibits villin-induced F-actin bundling. Thus villin expresses at least two distinct actin-binding sites localized on separate functional domains. Protein sequence analysis documents that the core comprises the NH2-terminal portion of intact villin, whereas the headpiece covers the COOH-terminal 76 amino acids. We provide the amino acid sequence of the headpiece, which is currently the smallest F-actin binding peptide.
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D005779:Immunodiffusion; D008840:Microfilament Proteins; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding
chemical_list: D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; C026615:villin